Histone methylation is one of several chromatin modifications that impact cellular processes such as transcription, development, and differentiation. The enzymes that regulate methylation, histone methyltransferases and demethylases, have been implicated in both disease (e.g. cancer and neurodegenerative disorders) as well as in promising therapeutic strategies (iPS reprogramming). Thus, developing small molecule probes that target these specific enzymes may have relevance for both basic research and clinical application. We propose to screen for inhibitors of the histone demethylase GASC-1 (gene amplified in squamous cell carcinoma-1), an enzyme with roles in cancer, androgen receptor signaling and the maintenance of pluripotency. GASC-1 directly and specifically removes repressive histone H3 lysine 9 tri-methylation in an iron and 2-oxoglutarate dependent hydroxylation mechanism. Currently, only general hydroxylase inhibitors are known to regulate histone demethylase activity, but no compounds specific for GASC-1 or even histone demethylases in general are available. To screen for such substances, we developed a biochemical assay for GASC-1 activity using a substrate peptide immobilized to streptavidin-coated 384-well plates and antibody-based detection of the demethylated product in a DELFIA assay. This assay is well established with an excellent Z'factor and extremely low rates of false-positive hits due to the direct detection mode. In a pilot screen, we tested ~28,000 compounds from the Broad Institute Screening Collection and obtained the first hit compounds. Furthermore, we established secondary cellular assays that monitor histone methylation changes following overexpression of the demethylase and GASC-1 dependent effects on proliferation and differentiation. PUBLIC HEALTH RELEVANCE: We intend to generate small-molecules specifically inhibiting histone demethylases and in particular GASC-1, an enzyme involved in cancer development and stem cell identity. These molecules will play important roles in basic research for understanding the contribution of histone methylation to epigenetic inheritance, but also have the potential to contribute to human health in the treatment of certain types of cancer and for applications in regenerative biology.